Rapid Embedding Methods into Epoxy and LR White Resins for Morphological and Immunological Analysis of Cryofixed Biological Specimens
Rapid Embedding Methods into Epoxy and LR White Resins for Morphological and Immunological Analysis of Cryofixed Biological Specimens
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DOI：
10.1017/s1431927613013846
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发表时间：
2014-02-01
影响因子：
2.8
通讯作者：
McDonald, Kent L.
中科院分区：
文献类型：
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作者：
McDonald, Kent L.
各种标本，包括细菌，纤毛虫，choanoflagellates（Salpingoeca rosetta），斑马鱼（Danio rerio）的胚胎，线虫（Caushabditis elegans），和叶的白色三叶草（白三叶）植物高压冷冻，冷冻取代，渗透与Epon，Epon-Araldite，或LR白色树脂，和聚合。从冷冻到切片的总处理时间约为6小时。对于环氧树脂包埋，将标本冷冻置换在1%四氧化锇加0.1%乙酸双氧铀的丙酮溶液中。对于LR白色包埋，冷冻置换介质为0.2%乙酸双氧铀的丙酮溶液。快速渗透通过离心通过增加树脂浓度，随后在100 ℃聚合1.5-2小时来实现。超微结构的保存与需要数天才能完成的标准冷冻置换和树脂包埋方法相当。肌动蛋白和微管蛋白分子的切片免疫标记结果为阳性，背景标记非常低。LR白色方法提供了一种更安全、更快速、更便宜的替代方法，可以替代Lowicryl包埋用于切片免疫标记的标本，而无需使用传统的醛类固定剂。
A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100 degrees C for 1.5-2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.